CD4+ T cell responses are thought to critical for the long term maintenance of effective host defense against chronic viral infections. Although animal models have been instrumental in delineating the general mechanisms underlying immunologic memory, detailed understanding of the development; functional heterogeneity, and homeostasis of antigen (Ag)-specific CD4+ memory cells in the human has not been accomplished. In this proposal, we will characterize fresh (e.g. non-cloned), viral pathogen-specific -- CMV, varicella zoster virus (VZV), and HIV - human CD4+ T cells with respect to the clonal complexity of their TCR (including characterization of dominant clonotypes), and their cytokine synthesis capabilities and activation threshold heterogeneity at the single cell and clonotype level. We bring to this effort novel technologies -- including 1) multiparameter flow cytometric analysis of Ag-specific CD4+ memory populations by detection of intracellular cytokine(s), CD40L and CD69 after short term activation with Ag, 2) characterization of clonotypic complexity and dominant clonotypes by PCR analysis of T cell receptor CDR3 regions on FACS-sorted Ag-specific CD4+ T cells, 3) quantification of clonotype +, Ag-specific, CD4+ T cells by QC-PCR, and 4) simultaneous analysis of TCR-Vbeta CDR3 and cytokine gene expression by single cell PCR -- that will enable us to functionally define and precisely quantify individual clonotypes among pathogen-specific human T cells, and follow the fate of these clonotypes with time, with exposure to Ag, or under conditions of T cell regeneration. Using these approaches we will investigate 1) mechanisms responsible for clonotypic dominance among CMV-, VZV-, and HIV-specific CD4+ memory T cells, 2) the extent and pattern of intra- and inter-clonal functional heterogeneity (cytokine synthesis and activation threshold) among these cells, 3) the degree of clonotypic expansion during the naive to memory transition, 4) pathways of differentiation within the memory subset (including the question of whether there is a true memory to naive "reversion"), and 5) the effect of time, Ag (re-exposure or elimination), progressive HIV immunodeficiency, and T cell regenerative mechanisms (after autologous stem cell transplantation and viral suppression in HIV disease) on the frequency and function of these viral-specific clonotypes. We are particularly interested in understanding the biology of HIV-specific CD4+ memory T cells, including the mechanisms responsible for their decline after long term viral suppression with effective antiretroviral therapy.